Association of ovarian response with picoAMH in women undergoing controlled ovarian hyperstimulation.

OBJECTIVE: Our study aims to evaluate the diagnostic performance of a high-sensitivity picoAnti-Müllerian Hormone (picoAMH) for predicting ovarian response in women undergoing controlled ovarian hyperstimulation and occurrence of ovarian hyperstimulation syndrome. METHODS: Retrospective cohort study at a single academic fertility center including all patients with picoAMH ELISA who underwent controlled ovarian hyperstimulation. The primary outcome was the number of oocytes retrieved, and secondary outcomes included cycle cancellation and ovarian hyperstimulation syndrome. Patients were grouped into poor, normal, and hyper-responders based on number of oocytes retrieved. RESULTS: The mean AMH and antral follicle count (AFC) were significantly different between normal response vs. hyper response group (p < 0.0001). Only serum AMH and not AFC was significantly increased in patients diagnosed with ovarian hyperstimulation syndrome (OHSS). For prediction of OHSS, receiver operating characteristic (ROC) analysis revealed that AMH (area under the ROC curve [AUC] = 0.85) was significantly better than the AFC (AUC = 0.64). The serum AMH cut-off at sensitivity of 80% for predicting OHSS among hyper responders from ROC curve was 3.67 ng/ml. Serum AMH measured by picoAMH ELISA showed superior correlation to number of oocytes retrieved when compared to AFC in the age group over 40 years old (r2 = 0.74 and r2 = 0.4, respectively) CONCLUSION: This study shows great utility of picoAMH ELISA for predicting ovarian response to controlled ovarian hyperstimulation (COH). Diagnostic performance of picoAMH for prediction of OHSS is superior to the AFC in our cohort.

Optimization of oestradiol assays to improve utility in an in vitro fertilization setting.

Background The measurement of oestradiol is an integral component for the management of ovarian stimulation for in vitro fertilization. Automated immunoassays offer fast assay times and high throughput, with less sensitivity and specificity. The aim of this study is to optimize the oestradiol assay in patients undergoing ovarian stimulation for in vitro fertilization via comparison of oestradiol values obtained using two immunoassays compared with mass spectrometry. Methods Patients undergoing ovarian stimulation were prospectively recruited. Serum samples were analysed with ADVIA Centaur® CP Immunoassay, Abbott Architect i1000® immunoassay and AB Sciex 5500 liquid chromatography-tandem mass spectrometry (LC-MS/MS) systems. Per cent bias was determined for each system to report the average tendency of the values to be larger or smaller than the LC-MS/MS value. Linear regression of total follicular volume and oestradiol was computed. Results The ADVIA Centaur® CP assay had a positive bias of 20% compared with LC-MS/MS, while the Architect i1000® had a non-significant, negative bias of 0.3%. With regression fit, a clear, positive relationship was seen between follicular volume and oestradiol. The Architect i1000® assay had a greater correlation (R2 = 0.46) compared with Centaur® CP (R2 = 0.36), when oestradiol values were >1000 pg/mL (3670 pmol/L). Conclusions The Abbott Architect i1000® oestradiol assay exhibits greater agreement with LC-MS/MS and exhibited better correlation to follicular volume when oestradiol values are >1000 pg/mL (3670 pmol/L), prompting a change in the clinic's oestradiol platform. Attention to assay quality assurance via LC-MS/MS can improve the oestradiol accuracy and permit more informed clinical decisions for improved patient outcomes.

Function and structure of cilia in the fallopian tube of an infertile woman with Kartagener's syndrome.

In Kartagener's syndrome (KS), primary defects of the ciliary axoneme cause dyskinetic ciliary motion. Because ciliary motion is an important factor in normal ovum transport, ciliary dyskinesia may cause infertility. On the other hand, the existence of some ciliary activity, albeit abnormal, may account for fertility in some women with KS. In this case study, an infertile woman diagnosed with KS had normal results in all usual infertility tests. Biopsies of tubal mucosa were obtained at laparoscopy for ovum recovery during an in-vitro fertilization cycle. Ciliary activity, measured by laser light-scattering spectroscopy, was detected in all tubal specimens; however the majority of regions sampled showed no activity. In active regions, beat frequency ranged from 5 to 10 Hz, approximately 30% of normal. Electron microscopy showed similar morphological defects in both tubal and nasal mucosa. The number of cilia per cell was approximately 20% of normal. The major ultrastructural abnormality of cilia was an absence of the central microtubules. The only demonstrable explanation for this patient's infertility was primary ciliary dyskinesia associated with KS.

Development of a human granulosa cell culture model with follicle stimulating hormone responsiveness.

In order to study the effects of follicle-stimulating hormone (FSH) on differentiation of granulosa cells, a well-defined and validated in-vitro culture system is indispensable. In this study, pooled follicular aspirates were stimulated in vitro with FSH and luteinizing hormone (LH) for 2, 4 and 6 days, either immediately after plating or after 7 days of preincubation. Cultures were assayed for progesterone and oestradiol production. Fresh cells displayed very high basal progesterone production which could be stimulated with LH but not FSH. After preincubation, addition of LH and FSH resulted in dose-dependent increases of progesterone and oestradiol. When cultured on human fibronectin-coated wells, similar basal but higher progesterone concentrations after stimulation were observed. In comparison with serum-free media, addition of Serum-Plus resulted in higher basal and stimulated progesterone concentration, possibly due to the presence of serum factors. This study demonstrates firstly that after 7 days preincubation, cultures gained responsiveness to FSH but remained responsive to LH during 4 days of stimulation. This suggests a persisting differentiated cell population in vitro. Secondly, the use of human fibronectin extracellular matrix and serum promotes steroid production, either due to factors promoting cell growth and function or to availability of steroid precursors. Therefore one has to be cautious with interpretation of data obtained from this widely used culture system, employing highly differentiated cells obtained after ovarian stimulation for in-vitro fertilization for study of local regulation of granulosa cell function.

Purification and characterization of a human follicular fluid lipid transfer protein that stimulates human sperm capacitation.

Identification of the mechanisms responsible for sperm capacitation has been an active area of research for nearly four decades. Changes in the lipid composition of the sperm membrane is one of the biochemical events that occurs during sperm capacitation. We have been studying physiological effectors of some of these changes and have identified lipid transfer activity in fractions of human follicular fluid that stimulates sperm penetration of zona-free hamster oocytes. We report here the purification of a lipid transfer protein by sequential chromatography from human follicular fluid. This protein was purified greater than 20,000-fold for lipid transfer activity and greater than 28,000-fold for sperm penetration-inducing activity. This 64,000 molecular weight protein has a pI of approximately 5.0 and shares physicochemical characteristics with the plasma lipid transfer protein, LTP-I. Antibodies to LTP-I also recognize this protein and depletion of LTP-I from human follicular fluid by immunoaffinity chromatography renders the follicular fluid incapable of stimulating sperm penetration. We conclude that purified LTP-I is able to stimulate human sperm capacitation and that LTP-I is a molecule responsible for this stimulation in follicular fluid.

Early luteal progesterone supplementation during in vitro fertilization cycles. A randomized trial.

The effects of early luteal phase progesterone (P) supplementation were studied in women with endogenous serum P levels less than or equal to 12 ng/mL prior to embryo transfer. From a total of 129 cycles that received the same ovarian hyperstimulation protocol, 72 cycles were characterized by levels less than 12 ng/mL on the day prior to embryo transfer. Of those women, 42 (group B) were started on P supplementation one day prior to embryo transfer, and 30 (group C) were started according to the standard protocol after embryo transfer. The clinical course and outcomes in both groups were compared with 57 cycles that had P levels greater than or equal to 12 ng/mL (group A). The early P supplementation in group B resulted in a transient increase in P levels on the morning of embryo transfer as compared with group C. It did not, however, approach the levels seen in group A, which had higher P levels from the early luteal phase through embryo transfer and more favorable oocyte recovery, fertilization and cleavage rates. We were unable to improve the clinical outcome in group B as compared with group C by providing earlier P supplementation.

Relationship of bacteriologic characteristics to semen indices in men attending an infertility clinic.

Bacteria can be isolated from most seminal fluid samples, but the significance of these microorganisms is uncertain because most men lack symptoms associated with bacterial infection of the reproductive tract. We obtained semen samples from 37 men attending a Special Infertility Clinic and assessed the relationship between seminal fluid microorganisms and seminal fluid analysis including sperm motility, morphology, and concentration; the numbers of polymorphonuclear leukocytes and other white blood cells; and the hamster zona-free oocyte sperm penetration assay. Aerobic and/or anaerobic bacteria were recovered from 36 of the 37 samples. One hundred eighty-eight isolates (113 aerobes, 74 anaerobes, and one yeast) were recovered, with a mean of 5.2 isolates per semen specimen. The microorganisms recovered from the samples included: coagulase-negative staphylococci (89%), viridans streptococci (65%), diphtheroids (86%), Peptostreptococcus sp (62%), Bacteroides sp (27%), Gardnerella vaginalis (19%), Lactobacillus sp (16%), Actinomyces sp (16%), Enterococcus (11%), and Veillonella (11%). Other microorganisms including group B streptococcus, Hemophilus, Escherichia coli, Mobiluncus, and Clostridium were each recovered from fewer than 10% of the specimens. When the microbiology of seminal fluid specimens with or without polymorphonuclear leukocytes was compared, the presence of polymorphonuclear leukocytes in the semen was not associated with the isolation of staphylococci (33 versus 25%), viridans streptococci (33 versus 28%), Bacteroides sp (17 versus 37%), or Peptostreptococcus (31 versus 33%) (P greater than .05 for each comparison). The proportion of semen samples yielding bacterial isolates was similar after categorization by normal motility (more than 60%), pyospermia (six or more leukocytes per 100 sperm), sperm concentration, morphology, and a normal sperm penetration assay (11% or more).(ABSTRACT TRUNCATED AT 250 WORDS)

Lipid transfer activity in human follicular fluid: relation to human sperm capacitation.

A potentially important event during sperm capacitation is the loss of sperm membrane cholesterol. Although the exact mechanisms mediating this loss are not known, albumin and high density lipoprotein have been proposed as lipid acceptors. The authors propose that lipid transfer may be involved in capacitation as a specific mediator in the sequence of events leading to sperm membrane cholesterol loss. We present the first direct evidence of lipid transfer activity (LTA) in human follicular fluid (HFF). The redistribution of 14C-cholesteryl ester among human plasma lipoproteins was used as a measure of LTA (% Transfer [%T]). The HFF was fractionated by S-300 gel filtration chromatography and assayed for LTA. Three peaks of activity were consistently eluted from the column. Each peak of LTA also stimulated human sperm to penetrate zona-free hamster oocytes after short capacitating incubations. The peak with highest LTA (12.75 +/- 1.11%T) with an Mr approximately 68,000, gave the greatest stimulation (penetration index, PI: 3.34 +/- 0.96 fold increase above control, n = 4). The HFF also showed a significant dose response for both LTA and PI, whereas bovine serum albumin did not. These results demonstrate the existence of LTA in HFF and suggest that a specific lipid transfer protein may have a role in human sperm capacitation or acrosome reaction.

The clinical relevance of sex selection techniques.

Clinical and laboratory attempts to alter the sex ratio require more complete and thorough study. Improved identification of Y-bearing sperm through chromosome evaluation rather than by F-body identification is critical to provide a more precise definition. The tentative conclusions stated below are based on an assessment of literature from which it is generally difficult to draw conclusions: 1. The timing of intercourse in relation to ovulation and subsequent fertilization appears to influence the sex ratio. More females are conceived when coitus occurs relatively close to ovulation, and more males are conceived when the sperm or egg is in the reproductive tract for a relatively longer time before conception. The influence of coital timing on the sex ratio is overall quite subtle and is not a practical method to alter the sex ratio for individual couples. 2. The use of ovulation-inducing medications slightly favors female offspring. A decrease in sex ratio of 5% to 10% has been shown in multiple studies. 3. Artificial insemination with fresh donor or homologous spermatozoa results in more male births with a reported 7% to 10% increase in the sex ratio. It appears that ovulation induction combined with artificial insemination cancels the respective influences of each on the sex ratio. 4. Sperm separation techniques using albumin (for selection of Y-bearing sperm) or Sephadex column filtration (for selection of X-bearing sperm) are the only techniques that have been reported to alter the sex ratio to a degree that is clinically relevant. Although clinical birth data are just beginning to accumulate, these methods appear to have a 70% to 80% success for selection of assumed Y-bearing sperm and a 75% to 80% success for selection of assumed X-bearing sperm. The validity of these results will remain questionable until fully detailed accounts are published and successfully repeated. Free-flow electrophoresis appears to achieve significant separation; however, the depressed postprocedure spermatozoa motility presently limits the usefulness of this procedure. 5. There is a potential to combine clinical and laboratory methods to maximize the efficiency of sex selection for interested couples. Modern methods to identify ovulation (e.g., urinary LH kits, ultrasonography) may help the timing of coitus for sex selection. Clomiphene citrate may enhance female sex preselection when Sephadex column filtration is also employed. 6. The priority of sex preselection in terms of medical, social, and demographic consideration remains to be determined. The avoidance of sex-linked genetic disorders is a reasonable and desirable goal.(ABSTRACT TRUNCATED AT 400 WORDS)

Transmission of genital herpes by donor insemination.

This report describes a donor in a therapeutic donor insemination program who asymptomatically acquired a primary herpes simplex virus type 2 (HSV-2) infection from his long-standing sexual partner. His fresh semen was used to inseminate two HSV-seronegative recipients; in one a primary HSV-2 infection developed, and in one it did not. Direct evidence of transmission from donor to recipient was documented by restriction enzyme analysis of the HSV-2 isolates obtained from the donor's semen and from the recipient's cervix. Because of the possibility of asymptomatic acquisition and transmission of HSV-2, semen donors and their sexual partners should undergo serologic screening for genital herpes using new, type-specific HSV serologic techniques.

An in vitro fertilization program using satellite physicians.

In order to provide an in vitro fertilization (IVF) service for a large geographic region, we developed the concept of Satellite IVF Centers. The goals of this program were as follows: 1) to facilitate patient participation by decreasing travel expenditures, time for screening appointments, and IVF cycle cancellations, and 2) to involve community physicians in a regionalized program. We established centers in nine cities within the Washington, Alaska, Montana, and Idaho region serviced by the University of Washington, and in Alberta, Canada. A 2-day training session was held to provide participating physicians with a basic knowledge of IVF. We considered the roles of the satellite IVF physician to include identification of couples for IVF, initiation of ovulation induction cycles, and determination of appropriate induction cycles for oocyte recovery. The staff of the University Department of Laboratory Medicine standardized the methodologies for estradiol measurements and maintained a quality control analysis for all participating community laboratories. Satellite activity was coordinated through the IVF office at the University, which monitored physicians' decisions based on hormone and ultrasound data from days 7 and 8 of the stimulation cycle. Patients meeting specific ovarian response criteria arrived at the University on day 9 of the stimulation cycle and completed IVF. During 18 months, 72 patients were screened and initiated cycles at the nine participating centers. Forty-four of these patients were sent to the University for oocyte recovery, which resulted in 39 embryo transfers and eight pregnancies. This service has reached a large geographic community, dramatically reduced the cost of IVF for this community, and met with both patient and physician acceptance.

Comparison of three wash techniques for the removal of microorganisms from semen.

The objective of this study was to compare the effectiveness of three sperm separation techniques for producing samples free of seminal fluid microbes. Each of 11 semen samples were separated by the following techniques: wash, with centrifugation only; swim-up, with undiluted semen layered beneath medium; and wash and swim-up, with centrifuged sperm cells overlain with medium. Microbiology for both aerobic and anaerobic organisms of semen and washes was determined by standard methods. The microbes isolated from semen were similar to those found in previous reports. However, the swim-up technique proved more successful than either the wash or the wash and swim-up methods for the recovery of sterile spermatozoa (ten of 11 versus four of 11, P less than .05). It is recommended that the swim-up technique, with medium supplemented with penicillin and streptomycin, be used when preparing sperm samples for placement into the female reproductive tract.

Human sperm penetration assay as an indicator of sperm function in human in vitro fertilization.

Performance of spermatozoa in a hamster oocyte/human sperm penetration assay (SPA) was correlated with the results of in vitro fertilization (IVF). Forty-two patients underwent 50 IVF cycles. SPA scores were obtained before IVF cycles (screening SPA, n = 30) and, where practical, on the semen sample used for IVF (IVF SPA, n = 26). Screening SPA score did not correlate to IVF result, fertilization and cleavage rates were similar between normal (n = 17) and low (n = 13) SPA groups, pregnancy rates were 35 and 46%, respectively. In addition, SPA score at the time of IVF did not correlate with IVF result. Pregnancy rates were 33% for the normal group (n = 16) and 30% for the low SPA group (n = 10). Overall, the low SPA group (n = 16) exhibited a 78% fertilization rate and a 38% pregnancy rate, which was not different from the normal SPA group: 76 and 29%, respectively. The results of this study indicate that SPA score is a poor indicator of sperm function in IVF.

Starfish oocyte maturation: 1-methyladenine triggers a drop of cAMP concentration related to the hormone-dependent period.

Oocyte maturation (meiosis reinitiation) in starfish is induced by the natural hormone 1-methyladenine (1-MeAde). Oocytes of Evasterias troschelii contain 0.43 pmole cyclic AMP/mg protein and 0.47 pmole cyclic GMP/mg protein. Upon stimulation by 1-MeAde the oocytes undergo a moderate (10-30%) decrease in their cAMP concentration. The concentration of cGMP remains unaltered. Oocytes treated with forskolin, an activator of adenylate cyclase, increase their cAMP concentration over 35-fold, up to 16 pmole cAMP/mg protein. When stimulated by 1-MeAde these forskolin-pretreated oocytes undergo a major (50-70%) decrease in their cAMP concentration. A similar decrease is triggered by mimetics of 1-MeAde, such as dithiothreitol, arachidonic acid (AA), and 8-hydroxyeicosatetraenoic acid (8-HETE), but not by adenine which is inactive. 1-MeAde-stimulated oocytes of Pisaster ochraceus also undergo a decrease in cAMP content, the size of which is increased by forskolin. Although a decrease in cAMP begins at sub-threshold 1-MeAde concentrations, the maximal decrease occurs at the same concentration of 1-MeAde needed for maturation induction and a further 1000-fold increase of the 1-MeAde concentration has no further effect. Upon removal of 1-MeAde, the cAMP concentration immediately increases to its original level. Sequential addition and removal of 1-MeAde triggers a sequential decrease and increase of the cAMP concentration, illustrating the continuous requirement for 1-MeAde for eliciting the decrease. Successive additions of 1-MeAde, however, do not trigger further decreases of the cAMP concentration. The temperature dependences of the cAMP concentration decrease and of the hormone-dependent period (HDP; the time of contact with 1-MeAde required for induction of maturation) are closely related. Forskolin, which increases the cAMP concentration, also increases the duration of the HDP (2.5-fold), delays the time course of protein phosphorylation burst and germinal vesicle breakdown, and inhibits AA- and 8-HETE-induced maturation. We conclude that 1-MeAde triggers a drop in cAMP concentration, which is tightly associated with the hormone-dependent period of oocyte maturation.

Ectopic pregnancy. Diagnosis by sonography correlated with quantitative HCG levels.

Pelvic sonograms were correlated with simultaneous human chorionic gonadotropin (HCG) determinations in 150 women with early intrauterine pregnancy (N = 76) and ectopic pregnancy (N = 74). Of the 76 patients with intrauterine pregnancy (IUP), 55 had HCG levels exceeding 1,800 mIU/ml (Second International Standard), and in each case a gestational sac was identified. In comparison, 35 of 74 (47%) patients with ectopic pregnancy had HCG levels of 1,800 mIU/ml or more, and no case demonstrated a gestational sac. Although six patients (8%) with ectopic pregnancy demonstrated a "pseudogestational sac," no case was confused with a true gestational sac. We conclude that, when the HCG level exceeds 1,800 mIU/ml, an intrauterine gestational sac is normally detected and its absence is evidence for an ectopic pregnancy.

Endocrine changes in the late-follicular and postovulatory intervals as determinants of the in vitro fertilization pregnancy rate.

This investigation examines the hormone pattern in in vitro fertilization (IVF) cycles from the time of human chorionic gonadotropin (hCG) administration through embryo transfer to ascertain whether the absolute levels or secretory patterns of the major reproductive hormones affect the IVF pregnancy rate. Thirty-one women who underwent IVF treatment were enrolled in the study. All patients received clomiphene citrate/human menopausal gonadotropin for ovulation induction. Significant elevations in serum estradiol (E2) levels in the pregnant group were found throughout the cycle interval studied. After hCG administration the serum hCG levels were not different between the groups. Significant elevations in serum progesterone (P) concentrations were found in the pregnant group from the day after laparoscopy through embryo transfer. Embryos obtained from the pregnant group appeared to be different in that the mean number of blastomeres per embryo transferred was significantly greater. Therefore for achievement of an IVF pregnancy the optimal hormone pattern employing combination ovulation induction in the ovulation to transfer interval is a relatively high E2 level in ovulation followed by a high P level at transfer and into the luteal phase. These elevated hormone levels do not depend on the response to exogenous hCG.

Amniotic membranes as a temporary wound dressing in necrotizing fasciitis.

A diabetic patient was seen at Magee-Womens Hospital with a vulvar abscess extending to the abdomen. Necrotizing fasciitis was diagnosed at operation, and the involved tissue was widely excised. The authors describe the successful use of amniotic membranes as a temporary wound dressing. The benefits of this method over conventional gauze dressings are easy availability, better adherence to the wound, less pain, and negligible cost to the patient.